Optimised helper virus-free production of high-quality adeno-associated virus vectors.

BACKGROUND: Clinical development of adeno-associated virus (AAV) requires standardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several strategies have been reported for the approach to the manufacturing problem. We report a helper virus-free process that produces high quality rAAV stocks. METHODS: rAAV were produced by triple transfection, a helper virus-free process. After lysis of the cells in the presence of nuclease, the rAAV produced were purified by HPLC through two ion-exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological activity and for the presence of residual contaminants. The titer of infectious particles and of rep + particles was determined by dRA assay. Contaminating DNA and RNA were determined by fluorescent dye binding and real-time PCR. The protein content of the rAAV stocks was characterised by SDS-PAGE, ELISA test, Western blot and specific enzymatic assays for putative residual contaminating protein. The in vivo biological activity of the stocks was evaluated in mouse muscle. RESULTS: rAAV stocks obtained following this procedure elicit: 2-5 x 10(12) pp/ml; 3-6 x 10(10) ip/ml; < 10(3) rep + particles/ml; <0.3 mUeq/ml of residual benzonase activity; non-detectable Ad or beta-galactosidase proteins; <35 pg/ml of cellular genomic DNA; in vivo expression in mouse muscle without any immune reaction detected. CONCLUSIONS: This work demonstrates the possibility of producing purified high-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors.

High throughput production, screening and analysis of adeno-associated viral vectors.

Recombinant adeno-associated viruses (rAAV) are promising candidates as gene vectors, as they transduce non-dividing cells and permit lasting transgene expression in a wide spectrum of tissues. In this paper, we describe a robust procedure for the high throughput production, screening and characterization of rAAV vectors. The technology includes the production of rAAV from rapid small scale plasmid preparations and the analysis of virus productivity (physical and infectious particles) and activity (transgene expression, replication). rAAV are produced by triple transfection (rAAV plasmid and AAV- and adenovirus (Ad)-helper plasmids) on 293 human embryo kidney (HEK) cells. The titers of physical and infectious particles are obtained by dot blot hybridization and by a serial dilution assay, followed by either dot blot hybridization or real-time PCR, respectively. rAAV can be produced and characterized from plasmid mixtures containing as little as 1/100 productive molecules. Experiments on rAAV replication kinetics and Ad helper functions are discussed. All steps are performed in 96-well microtiter plates. The process is reproducible, high throughput, linear and ready for automation.